title: Cloning, expression and functional analysis of PKR from grass carp (Ctenopharyngodon idellus).
abstract: The interferon-induced, dsRNA-activated protein kinase (PKR) is considered as an important component of innate immune system and as a representative effector protein of interferon system. In the present study, PKR gene (CiPKR, JX511974) from grass carp (Ctenopharyngodon idellus) was isolated and identified using homology-based PCR. CiPKR shares high sequence identity with the counterparts of goldfish (Crucian carp) and zebrafish (Danio rerio). The full-length cDNA of CiPKR was found to be 2436 bp, with an ORF of 2067 bp that encodes a polypeptide of 688 amino acids. The deduced polypeptide CiPKR contains three tandem dsRNA-binding motifs (dsRBMs) at the N-terminus and a conserved Ser/Thr kinase domain at the C-terminus. CiPKR was expressed ubiquitously at a low-level under normal conditions, but it could be up-regulated after intraperitoneal (ip) injection with grass carp haemorrhagic virus (GCHV). CiPKR was dramatically up-regulated at 6 h post-injection and then recovered rapidly to normal levels within 24 h; however, it was obviously up-regulated once again at 48 h or 72 h post-injection. It seemed that CiPKR could respond to GCHV infection in an IFN-independent as well as an IFN-dependent pathway. To further investigate its mechanism of biological actions, we constructed a series of recombinant plasmids including pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C (deletion of dsRBD sequence) and pcDNA3.1/PKR-C-K430R, and then each recombinant plasmid was transfected into CIK cells. In comparison with those of controls, a 79% and a 64% decrease of luciferase activities were detected in the tested cells transfected with CiPKR and CiPKR-C, respectively; however, luciferase activities were increased in those cells transfected with PKR-K430R and PKR-C-K430R, with a 160% and 115% up-regulation, respectively. Similarly, MTT colorimetric assay indicated that cell viabilities of CIK cells transfected with pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C and pcDNA3.1/PKR-C-K430R were 49%, 90%, 54% and 100%, respectively. Our observations suggested that the expression of CiPKR could be up-regulated following viral infection, and then resulted in the inhibition of protein synthesis and the induction of potential apoptosis.
authors.affiliation: Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China; Medical College, Jinggangshan University, Ji'an 343009, China.
authors.forename: You-Sheng
authors.initials: YS
authors.lastname: Hu
authors.forename: Wen
authors.initials: W
authors.lastname: Li
authors.forename: Dong-Ming
authors.initials: DM
authors.lastname: Li
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Fan
authors.forename: Ze-Chang
authors.initials: ZC
authors.lastname: Rao
authors.forename: Gang
authors.initials: G
authors.lastname: Lin
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D055612:Cytokine-Induced Killer Cells / Q000166:cytology / Q000821:virology; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012087:Reoviridae; D019892:eIF-2 Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D012333:RNA, Messenger; D019892:eIF-2 Kinase
keywords: Antiviral activity; CiPKR; Grass carp; Interferon; dsRNA